We have established conditions for in vitro assembly of stable synaptic complexes of a pair of viral DNA ends with HIV-1 integrase. These nucleoprotein complexes are intermediates in the integration of HIV DNA into a target DNA. Furthermore, the association of integrase with viral DNA in these complexes mimics all the properties of the association of integrase with viral DNA in preintegration complexes (PICs) isolated from virus infected cells. The synaptic complexes contain a tetramer of integrase tightly bounds to a pair of viral DNA ends. Footprinting of the viral DNA ends within the complex reveals that less than 20 base pairs of terminal viral DNA sequence are protected by integrase. In order to gain insight of the organization of two DNA segments within the SSC, we have investigated the SSC assembly process by fluorescence resonance energy transfer (FRET). Cy3 and Cy5 fluorophores have been incorporated at various positions along the DNA substrate and the FRET signal measured upon assembly into the SSC. The results show a significant FRET signal in the SSC when fluorophores are positioned close to the ends of the viral DNA. FRET efficiency was reduced when the fluorophores were located away from the end. The results suggest that the two DNA segments are anti-parallel within the SSC, a conclusion that is also in agreement with atomic force microscope images of the stable nucleoprotein complexes. 20bp of terminal viral DNA end sequence are efficient substrates for half-site integration in vitro, and are the only protected region observed in footprinting experiments. However, several hundred base pairs of non-specific flanking DNA sequence are required for efficient SSC assembly, stability and concerted integration. We are probing the function of this non-specific DNA sequence in SSC assembly and stability and propose that non-specific interactions between IN and DNA (distinct from the stable association of a tetramer of IN with the viral DNA ends) are involved. The potential role of cellular proteins in SSC assembly have also been investigated. One cellular protein that has been implicated in playing an important role in HIV-1 DNA integration is Lens Epithelial Derived Growth Factor (LEDGF). We find that LEDGF does not stimulate assembly of the SSC and in fact inhibits complex assembly. LEDGF must therefore be acquired by the preintegration complex after the two viral DNA ends are engaged by integrase to form the SSC.